Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon epithelium in toxicological studies in vitro.
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PMID:Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes. 1096 60

Fluorescence microscopy was used to visualize the accumulated fluorescent product of the enzyme alkaline phosphatase to indicate where active covalently bound enzyme remained on the surface after application of a Nd: YAG laser interference pattern to a surface that was first globally derivatized with the covalently bound enzyme. The electrochemical kinetics of the same carbon fiber surface were examined through the electrogenerated chemiluminescence of Ru(bpy)(3)2+ to determine that electron-transfer sites were indeed segregated from the enzyme-binding sites. The enzyme-derivatized areas are determined to be separate and distinct from the areas of enhanced electron transfer. Two other enzymes, glucose oxidase and malic dehydrogenase, were then covalently bound to carbon fiber microelectrode surfaces in order to verify the change in detection limit of their respective cofactors, NADH or H2O2, under a variety of surface conditions. The S/N of an enzyme-modified electrode after laser interference pattern photoablation and electrocatalytic treatment is improved by more than 1 order of magnitude over that observed at an electrode that is globally enzyme modified.
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PMID:Laser interference pattern ablation of a carbon fiber microelectrode: biosensor signal enhancement after enzyme attachment. 1105 9

NADH:quinone oxidoreductase, or Complex I, is a multi-subunit membrane-bound enzyme in the respiratory chain of many pro- and eukaryotes. The enzyme catalyzes the oxidation of NADH and donates electrons to the quinone pool, coupled to proton translocation across the membrane, but the mechanism of energy transduction is not understood. In bacteria the enzyme consists of 14 subunits, seven membrane spanning and seven protruding from the membrane. The hydrophobic NuoH (NQO8, ND1, NAD1, NdhA) subunit is seemingly involved in quinone binding. A homologous, structurally and most likely functionally similar subunit is also found in F(420)H2 oxidoreductases and in complex membrane-bound hydrogenases. We have made theoretical analyses of NuoH and NuoH-like polypeptides and experimentally analyzed the transmembrane topology of the NuoH subunit from Rhodobacter capsulatus by constructing and analyzing alkaline phosphatase fusion proteins. This demonstrated that the NuoH polypeptide has eight transmembrane segments, and four highly conserved hydrophilic sequence motifs facing the inside, bacterial cytoplasm. The N-terminal and C-terminal ends are located on the outside of the membrane. A topology model of NuoH based on these results is presented, and implications from the model are discussed.
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PMID:Transmembrane orientation and topology of the NADH:quinone oxidoreductase putative quinone binding subunit NuoH. 1124 99

CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the native phoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell extract and whole-cell activity studies showed that the periplasmically located CYP105D1 competently catalyzed NADH-dependent oxidation of the xenobiotic compounds benzo[a]pyrene and erythromycin, further revealing the presence in the E. coli periplasm of endogenous functional redox partners. This system offers substantial advantages for the application of P450 enzymes to whole-cell biotransformation strategies, where the ability of cells to take up substrates or discard products may be limited.
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PMID:Export of cytochrome P450 105D1 to the periplasmic space of Escherichia coli. 1131 92

Von Ebner's gland of ferret was examined by means of light microscopy, protein, mucosubstance and enzyme histochemistry, and neurohistology. Acinar cells were replete with granules containing neutral mucosubstances and disulphides, and showed strong diffuse acid phosphatase activity and weak granular staining for peroxidase. Staining for cytochrome oxidase, succinate dehydrogenase, and NADH and NAD(P)H dehydrogenases was also seen. Basolateral plasmalemma of acinar cells showed weak, ouabain-sensitive Na+,K+-ATPase activity. Ductal cells were of a simple appearance, contained thiols and showed variable staining for acid phosphatase, dehydrogenases and cytochrome oxidase. Variable amounts of beta-glucuronidase reaction product were localized in the glandular parenchyma, being marked in atrophic areas. Prominent stellate myoepithelial cells embracing acini and also basal ductal cells were demonstrated by alkaline phosphatase. Thiamine pyrophosphatase reaction product was concentrated in blood vessels around parenchyma, with little Golgi-like staining in acinar cells. Acetylcholinesterase activity was associated with an extensive network of nerve fibres embracing parenchyma, whereas catecholamine fluorescence was not seen. The results suggest that the acini of von Ebner's gland of ferret synthesise neutral secretory glycoproteins and peroxidase. Water mobilization is inconspicuous. Lysosomal activities feature in the parenchyma, possibly a consequence of processing secretory products in acini, absorption in ducts and/or adaptation atrophy. The gland receives a rich cholinergic-type innervation, and has extensive myoepithelial and microvascular networks.
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PMID:Histochemical phenotypes of von Ebner's gland of ferret and their functional implications. 1150 41

A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.
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PMID:Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay. 1175 40

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.
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PMID:Oxidative DNA damage induced by equine estrogen metabolites: role of estrogen receptor alpha. 1195 37

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called "occluded" intermediary state of the reaction cycle of the intact enzyme. Despite a K(d) in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP(+) is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2'-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD(+) by NADH, indicating that 3-acetylpyridine-NAD(+) and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2'-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.
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PMID:Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme. 1276 62

This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H(2)NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds.
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PMID:Structure and dynamics of proteins encapsulated in silica hydrogels by Trp phosphorescence. 1283 35

Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were investigated. The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+). Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+). The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate. This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase.
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PMID:A spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase. 1535 57


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